1. Field of the Invention
The present invention relates to the Trypanosoma cruzi proline racemase, a 45 kDa polyclonal activator. More specifically, the present invention relates to the crystal structure of the TcPA45 (TcPRAC) protein of T. cruzi, methods of obtaining crystals and crystal structures of the TcPA45 (TcPRAC) protein of T. cruzi, and methods of using the crystal structure of the TcPA45 (TcPRAC) protein of T. cruzi to identify drugs that affect the pathogenicity of T. cruzi. 
2. Description of the Related Art
D-amino acids have long been described in the cell wall of eubacteria, where they constitute essential elements of the peptidoglycan and act as substitutes of cell wall teichoic acids (4), and in other parts of eubacteria as part of small peptides made by non-ribosomal protein synthesis (3, 4). In contrast, until recently it was believed that only L-amino acid enantiomers were present in eukaryotes (apart from a very low level of D-amino acids from spontaneous racemization due to aging) (1). However, recently an increasing number of studies have reported the presence of various D-amino acids (D-aa) in both protein-bound (5) and free forms (6) in a wide variety of eukaryotes, including mammals. The origin of free D-aa can be exogenous (9) or endogenous (7, 8, 10-12) to the eukaryote.
Proline racemase catalyzes the interconversion of L- and D-proline enantiomers, and has, to date, been described in only two species, Clostridium sticklandii and Trypanosoma cruzi. The enzyme from the eubacterium C. sticklandii contains cysteine residues in the active site, and does not require co-factors or known co-enzymes for activity. As disclosed in U.S. Provisional Patent Application No. 60/446,263, U.S. patent application Ser. No. 09/725,945 (now U.S. Pat. No. 6,713,617) and in Chamond N. et al (J. Biol. Chem., 2003, 278, 18, 15484-15494), the enzyme from the parasitic eukaryote T. cruzi, which causes Chagas' Disease in humans, exists in two forms, TcPRACA and TcPRACB, encoded by two independent genes, respectively. The T. cruzi TcPRACB enzyme represents an intracellular form of the enzyme that is present in non-infective forms of the organism. The T. cruzi TcPRACA enzyme represents a membrane-bound or secreted form of the enzyme that is present in infective forms of the organism. TcPRACA may also originate an intracellular version of proline racemase by a mechanism of alternative splicing. The two forms of the enzyme share a high level of homology, and appear to be a result of gene duplication. A cysteine at residue 330 of the TcPRACA enzyme is located in the active site of the enzyme. A cysteine at position 160 of the TcPRACA is also involved in the active site of the enzyme. The TcPRACA enzyme is a potent host B-cell mitogen that supports parasite evasion of specific immune responses, and has been implicated in persistence of the parasite through polyclonal lymphocyte activation (10). The mitogenic properties of the T. cruzi proline racemase are dependent on the integrity of the enzyme active site (2).
In view of the importance of both forms of the TcPRAC enzyme (i.e., TcPRACA and TcPRACB) to the growth and infectivity of T. cruzi, structural and biochemical information on the enzyme is needed to provide new drugs and methods for treating T. cruzi infection.